Abstract

Background

It is thought that foamy viruses (FVs) enter host cells via endocytosis because all
FV glycoproteins examined display pH-dependent fusion activities. Only the prototype
FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting
that its uptake mechanism may deviate from other FVs. To gain new insights into the
uptake processes of FV in individual live host cells, we developed fluorescently labeled
infectious FVs.

Results

N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein
resulted in efficient incorporation of the fluorescently labeled glycoprotein into
secreted virions without interfering with their infectivity. Double-tagged viruses
consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env)
from either PFV or macaque simian FV (SFVmac) were observed during early stages of
the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong
syncytia formation on target cells. Both virus types showed trafficking of double-tagged
virions towards the cell center. Upon fusion and subsequent capsid release into the
cytosol, accumulation of naked capsid proteins was observed within four hours in the
perinuclear region, presumably representing the centrosomes. Interestingly, virions
harboring fusion-defective glycoproteins still promoted virus attachment and uptake,
but failed to show syncytia formation and perinuclear capsid accumulation. Biochemical
and initial imaging analysis indicated that productive fusion events occur predominantly
within 4–6 h after virus attachment. Non-fused or non-fusogenic viruses are rapidly
cleared from the cells by putative lysosomal degradation. Quantitative monitoring
of the fraction of individual viruses containing both Env and capsid signals as a
function of time demonstrated that PFV virions fused within the first few minutes,
whereas fusion of SFVmac virions was less pronounced and observed over the entire
90 minutes measured.

Conclusions

The characterized double-labeled FVs described here provide new mechanistic insights
into FV early entry steps, demonstrating that productive viral fusion occurs early
after target cell attachment and uptake. The analysis highlights apparent differences
in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled
FVs promise to provide important tools for future detailed analyses on individual
FV fusion events in real time using advanced imaging techniques.